Synthesis of 1,25-dihydroxyvitamin D in human bladder epithelial cells and vitamin D mediated cathelicidin induction
Jeanette Bennett1, Daniel Zehnder1,2, Kieran Jefferson2 & Rosemary Bland1
It is now recognized that vitamin D influences the innate immune system and recent studies have highlighted the importance of local synthesis of 1,25-dihydroxyvitamin D (1,25D) for these responses. Production of 1,25D from 25-hydroxvitamin D (25D) is catalyzed by 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1; 1α-OHase). Vitamin D is metabolized by 24-hydroxylase (CYP24A1; 24-OHase). A key part of the innate immune response is pathogen recognition by the toll-like receptors (TLR) and an important immune target gene is cathelicidin (LL37), a vitamin D responsive antimicrobial peptide. This study examined the expression of vitamin D and TLR signaling components, the synthesis of 1,25D and the induction of cathelicidin by vitamin D in bladder epithelial cells. Studies used human bladder epithelial (urothelium) cell lines (T24/83 and RT4) derived from transitional cell carcinomas. Both cell lines expressed the VDR and 1α-OHase mRNA (RT-PCR) and protein (Western blot analysis). In addition, the cells expressed RXRα, β and γ and megalin mRNA. 24-OHase mRNA, which was almost undetectable in unstimulated cells, was significantly increased by 1,25D (10 nM; 3-24 h; P<0.05). 24-OHase activity was confirmed. 24-OHase mRNA was also significantly increased by 25D (100 nM; 6-24 h; P<0.05) indicating epithelial 1α-OHase activity. Synthesis of 1,25D was confirmed by ELISA. Bladder cells produced 1,25D at levels similar to HKC-8 cells (a human kidney cell line) and synthesis was attenuated following pre-treatment with ketoconazole (2 h, 10 μM). Both cell types expressed TLR 1, 2 & 4 mRNA and the TLR partners MyD88 and CD14. Cathelicindin mRNA was undetectable in both cell lines, but was induced by 1,25D in RT4 cells (10 nM; 6-24 h; P<0.05). It was also increased by 25D (100 nM; 6 h; P<0.05). These data demonstrate that bladder epithelial cells express functional vitamin D signaling and are able to synthesize sufficient 1,25D to stimulate a local immune response.